P750: A ZEBRAFISH MODEL FOR JAGN1-ASSOCIATED SEVERE CONGENITAL NEUTROPENIA
Authors: Larissa Doll, Narges Aghaallaei, Claudia Lengerke, Cornelia Zeidler, Karl Welte, Julia Skokowa, Baubak Bajoghli
Journal: HemaSphere (2023)

Topic: 11. Bone marrow failure syndromes incl. PNH - Biology & Translational Research Background: Severe congenital neutropenia (CN) associated with autosomal recessive JAGN1 (Jagunal Homolog 1) mutations is a bone marrow failure syndrome characterized by a severely reduced number of neutrophils and poor response to G-CSF therapy. The discovery of an alternative or synergistic treatment for JAGN1-CN patients is highly important. However, the underlying pathomechanism of neutropenia downstream of JAGN1 mutations is not entirely elucidated yet, and there is no suitable in vivo model available. Jagn1 knock-out mice are embryonically lethal and hematopoietic lineage-specific deletion of Jagn1 does not induce CN in mice. Zebrafish (ZF) is a promising in vivo vertebrate model for human diseases - molecular and cellular mechanisms of hematopoiesis are largely conserved between ZF and human. Additionally, a large number of offspring and cell-specific fluorescent reporter lines are ideal requirements for the discovery of new drug candidates. Aims: Establish a ZF model to study the pathomechanism of JAGN1-CN and potentially discover new therapeutic strategies for this syndrome. Methods: To identify the functionally-related paralogue gene of human JAGN1 in ZF, we analyzed the expression pattern of jagn1a and b, using publicly available RNAseq databases. Jagn1b, but not jagn1a, is expressed in hematopoietic cells. Therefore, we interfered with the function of jagn1b to establish a JAGN1-deficient ZF model. We applied two different approaches: morpholino-based knock-down (KD) and CRISPR/Cas9-based knock-out (KO). The effects on hematopoiesis were analyzed by evaluating the expression patterns of mRNAs specific for neutrophils, HSPCs, erythrocytes, or macrophages using whole mount in situ hybridization (WISH). The importance of jagn1b in the induction of unfolded protein response (UPR) and apoptosis was analyzed using RT-qPCR and TUNEL assay, respectively. To elucidate the effect of UPR and apoptosis induction on the neutrophil numbers, we treated embryos with Tunicamycin (TUN), DTT or Staurosporin (STS), respectively and quantified the neutrophils with WISH. Moreover, the localization of Jagn1b protein was analyzed by imaging after injection of gfp tagged wild-type (wt) or mutated jagn1b mRNA into the Tg(drl:rfp) reporter, where HSPCs express rfp. Results: After interfering with the function of jagn1b, independent of the strategy, the numbers of neutrophils were significantly reduced without affecting HSPCs, macrophages, or erythrocytes, when compared to uninjected siblings. Furthermore, the expression levels of atf4a, atf4b and chop increased in jagn1b KD embryos, when compared to wt, showing that jagn1b KD induced UPR. Apoptosis was also elevated in jagn1b KD embryos compared to wt littermates, but not restricted to the hematopoietic tissues. Interestingly, UPR or apoptosis induction in wt and jagn1b KD embryos by TUN and DTT, or STS treatment respectively, did not affect the neutrophil numbers when compared to the untreated siblings. Taken together, neither UPR nor apoptosis activation alone is the cause of neutropenia, additional pathological events downstream of JAGN1 mutations are necessary to block granulopoiesis. Another interesting observation was the mislocalization of mutated jagn1b in the HSPCs of Tg(drl:rfp) after mRNA injection. Thus, wt Jagn1b was localized in an ER-structure-like pattern, but mutated was distributed randomly. The exact localizations need to be further elucidated. Summary/Conclusion: Overall, we have established an in vivo model for studying the role of JAGN1 in granulopoiesis which is a valuable tool for discovering new targets and therapeutic strategies for JAGN1-CN. Keywords: Animal model, Severe congenital neutropenia, Zebra fish